Hormones and polypeptide growth factors control proliferation and differentiation of cells of multicellular organisms. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.
Hormones and growth factors influence cellular metabolism by binding to receptors. Receptors may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble molecules, such as the transcription factors. Of particular interest are receptors for cytokines, molecules that promote the proliferation and/or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia, thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.
The demonstrated in vivo activities of these cytokines illustrate the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists or binding partners. The present invention addresses these needs by providing a new cytokine antagonist or binding partner, a soluble hematopoietic cytokine receptor, as well as related compositions and methods.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect, the present invention provides an isolated polynucleotide that encodes a soluble receptor polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence as shown in SEQ ID NO:6, and wherein the soluble receptor polypeptide encoded by the polynucleotide sequence binds a ligand comprising a polypeptide of SEQ ID NO:10 or SEQ ID NO:47, or antagonizes the ligand activity. In one embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a homodimeric receptor complex.
Within another aspect, the present invention provides an isolated polynucleotide that encodes a soluble receptor polypeptide comprising a sequence of amino acid residues that is at least 90% identical to the amino acid sequence as shown in SEQ ID NO:6, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex further comprising a soluble Class I cytokine receptor.
In one embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex further comprising a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82). In another embodiment, the isolated polynucleotide is as disclosed above, wherein the polypeptide further comprises a WSXWS motif as shown in SEQ ID NO:13.
Within another aspect, the present invention provides an isolated polynucleotide that encodes a soluble receptor polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:6, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide encoded by the polynucleotide further comprises a soluble Class I cytokine receptor. In another embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide encoded by the polynucleotide forms a heterodimeric or multimeric receptor complex further comprising a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82). In another embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide is encoded by the polynucleotide as shown in SEQ ID NO:7. In another embodiment, the isolated polynucleotide is as disclosed above, wherein the soluble receptor polypeptide further comprises an affinity tag.
Within a second aspect, the present invention provides an expression vector comprising the following operably linked elements: (a) a transcription promoter; a first DNA segment encoding a soluble receptor polypeptide having an amino acid sequence as shown in SEQ ID NO:6; and a transcription terminator; and (b) a second transcription promoter; a second DNA segment encoding a soluble Class I cytokine receptor polypeptide; and a transcription terminator; and wherein the first and second DNA segments are contained within a single expression vector or are contained within independent expression vectors. In one embodiment, the expression vector disclosed above further comprises a secretory signal sequence operably linked to the first and second DNA segments. In another embodiment, the expression vector is as disclosed above, wherein the second DNA segment encodes a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82).
Within a third aspect, the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses the polypeptides encoded by the DNA segments. In one embodiment, the cultured cell comprising an expression vector is as disclosed above, wherein the first and second DNA segments are located on independent expression vectors and are co-transfected into the cell, and cell expresses the polypeptides encoded by the DNA segments. In another embodiment, the cultured cell comprising an expression vector is as disclosed above, wherein the cell expresses a heterodimeric or multimeric soluble receptor polypeptide encoded by the DNA segments. In another embodiment, the cultured cell comprising an expression vector is as disclosed above, wherein the cell secretes a soluble receptor polypeptide heterodimer or multimeric complex. In another embodiment, the cultured cell comprising an expression vector is as disclosed above, wherein the cell secretes a soluble receptor polypeptide heterodimer or multimeric complex that binds a ligand comprising a polypeptide of SEQ ID NO:10 or SEQ ID NO:47, or antagonizes the ligand activity.
Within another aspect, the present invention provides a DNA construct encoding a fusion protein comprising: a first DNA segment encoding a polypeptide having a sequence of amino acid residues as shown in SEQ ID NO:6; and at least one other DNA segment encoding a soluble Class I cytokine receptor polypeptide, wherein the first and other DNA segments are connected in-frame; and wherein the first and other DNA segments encode the fusion protein. In one embodiment, the DNA construct encodes a fusion protein as disclosed above, wherein at least one other DNA segment encodes a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82).
Within another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA construct encoding a fusion protein as disclosed above; and a transcription terminator, wherein the promoter is operably linked to the DNA construct, and the DNA construct is operably linked to the transcription terminator.
Within another aspect, the present invention provides a cultured cell comprising an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA construct.
Within another aspect, the present invention provides a method of producing a fusion protein comprising:culturing a cell as disclosed above; and isolating the polypeptide produced by the cell.
Within another aspect, the present invention provides an isolated soluble receptor polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence as shown in SEQ ID NO:6, and wherein the soluble receptor polypeptide binds a ligand comprising a polypeptide of SEQ ID NO:10 or SEQ ID NO:47, or antagonizes the ligand activity. In one embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide forms a homodimeric receptor complex.
Within another aspect, the present invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence as shown in SEQ ID NO:6, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex further comprising a soluble Class I cytokine receptor. In another embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex further comprising a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82). In another embodiment, the isolated polypeptide is as disclosed above, wherein the polypeptide further comprises a WSXWS motif as shown in SEQ ID NO:13.
Within another aspect, the present invention provides an isolated soluble receptor polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:6, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex. In one embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex further comprising a soluble Class I cytokine receptor. In another embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide forms a heterodimeric or multimeric receptor complex comprising a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82). In another embodiment, the isolated polypeptide is as disclosed above, wherein the soluble receptor polypeptide further comprises an affinity tag, chemical moiety, toxin, or label.
Within another aspect, the present invention provides an isolated heterodimeric or multimetric soluble receptor complex comprising soluble receptor subunits, wherein at least one of soluble receptor subunits comprises a soluble receptor polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:6. In one embodiment, the isolated heterodimeric or multimetric soluble receptor complex disclosed above further comprises a soluble Class I cytokine receptor polypeptide. In another embodiment, the isolated heterodimeric or multimetric soluble receptor complex disclosed above further comprises a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4) or a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82).
Within another aspect, the present invention provides a method of producing a soluble receptor polypeptide that form a heterodimeric or multimeric complex comprising: culturing a cell as disclosed above; and isolating the soluble receptor polypeptides produced by the cell.
Within another aspect, the present invention provides a method of producing an antibody to a soluble receptor polypeptide comprising: inoculating an animal with a soluble receptor polypeptide complex selected from the group consisting of: (a) a polypeptide comprising a homodimeric soluble receptor complex comprising SEQ ID NO:6; (b) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO:6; (b) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO:6, and further comprising a soluble Class I cytokine receptor polypeptide; (c) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO:6, and further comprising a soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4); (d) a polypeptide comprising a soluble receptor heterodimeric or multimeric receptor complex comprising SEQ ID NO:6, and further comprising a soluble IL-13xcex1xe2x80x2 receptor polypeptide (SEQ ID NO:82); and wherein the polypeptide complex elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
Within another aspect, the present invention provides an antibody produced by the method as disclosed above, which specifically binds to a homodimeric, heterodimeric or multimeric receptor complex comprising a soluble receptor polypeptide comprising SEQ ID NO:6. In one embodiment the antibody disclosed above is a monoclonal antibody.
Within another aspect, the present invention provides an antibody which specifically binds to a homodimeric, heterodimeric or multimeric receptor complex as disclosed above.
Within another aspect, the present invention provides a method for inhibiting a ligand comprising a polypeptide of SEQ ID NO:10 or SEQ ID NO:47, or antagonizing the ligand activity-induced proliferation of hematopoietic cells and hematopoietic cell progenitors comprising culturing bone marrow or peripheral blood cells with a composition comprising an amount of soluble receptor comprising SEQ ID NO:6 sufficient to reduce proliferation of the hematopoietic cells in the bone marrow or peripheral blood cells as compared to bone marrow or peripheral blood cells cultured in the absence of soluble receptor. In one embodiment the method is as disclosed above, wherein the hematopoietic cells and hematopoietic progenitor cells are lymphoid cells. In another embodiment the method is as disclosed above, wherein the lymphoid cells are NK cells or cytotoxic T cells.
Within another aspect, the present invention provides a method of reducing proliferation of neoplastic B or T cells comprising administering to a mammal with a B or T cell neoplasm an amount of a composition of soluble receptor comprising SEQ ID NO:6 sufficient to reduce proliferation of the neoplastic B or T cells.
Within another aspect, the present invention provides a method of suppressing an immune response in a mammal exposed to an antigen or pathogen comprising: (1) determining a level of an antigen- or pathogen-specific antibody; (2) administering a composition of soluble receptor polypeptide comprising SEQ ID NO:6 in an acceptable pharmaceutical vehicle; (3) determining a post administration level of antigen- or pathogen-specific antibody; (4) comparing the level of antibody in step (1) to the level of antibody in step (3), wherein a lack of increase or a decrease in antibody level is indicative of suppressing an immune response.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:
The term xe2x80x9caffinity tagxe2x80x9d is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag(trademark) peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).
The terms xe2x80x9camino-terminalxe2x80x9d and xe2x80x9ccarboxyl-terminalxe2x80x9d are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
The term xe2x80x9ccomplement/anti-complement pairxe2x80x9d denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of  less than 109 Mxe2x88x921.
The term xe2x80x9ccomplements of a polynucleotide moleculexe2x80x9d is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5xe2x80x2 ATGCACGGG 3xe2x80x2 is complementary to 5xe2x80x2 CCCGTGCAT 3xe2x80x2.
The term xe2x80x9cdegenerate nucleotide sequencexe2x80x9d denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
The term xe2x80x9cexpression vectorxe2x80x9d is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
The term xe2x80x9cisolatedxe2x80x9d, when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5xe2x80x2 and 3xe2x80x2 untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
An xe2x80x9cisolatedxe2x80x9d polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term xe2x80x9cisolatedxe2x80x9d does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
The term xe2x80x9coperably linkedxe2x80x9d, when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
The term xe2x80x9corthologxe2x80x9d denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
xe2x80x9cParalogsxe2x80x9d are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, xcex1-globin, xcex2-globin, and myoglobin are paralogs of each other.
A xe2x80x9cpolynucleotidexe2x80x9d is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5xe2x80x2 to the 3xe2x80x2 end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated xe2x80x9cbpxe2x80x9d), nucleotides (xe2x80x9cntxe2x80x9d), or kilobases (xe2x80x9ckbxe2x80x9d). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term xe2x80x9cbase pairsxe2x80x9d. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired.
A xe2x80x9cpolypeptidexe2x80x9d is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as xe2x80x9cpeptidesxe2x80x9d.
xe2x80x9cProbes and/or primersxe2x80x9d as used herein can be RNA or DNA. DNA can be either cDNA or genomic DNA. Polynucleotide probes and primers are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., using techniques that are well known in the art.
The term xe2x80x9cpromoterxe2x80x9d is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5xe2x80x2 non-coding regions of genes.
A xe2x80x9cproteinxe2x80x9d is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The term xe2x80x9creceptorxe2x80x9d is used herein to denote a cell-associated protein, or a polypeptide subunit of such a protein, that binds to a bioactive molecule (the xe2x80x9cligandxe2x80x9d) and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a conformational change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain(s) and other molecule(s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. Cell-surface cytokine receptors are characterized by a multi-domain structure as discussed in more detail below. These receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues (typically about 21-25 residues), which is commonly flanked by positively charged residues (Lys or Arg). In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor). The term xe2x80x9creceptor polypeptidexe2x80x9d is used to denote complete receptor polypeptide chains and portions thereof, including isolated functional domains (e.g., ligand-binding domains).
A xe2x80x9csecretory signal sequencexe2x80x9d is a DNA sequence that encodes a polypeptide (a xe2x80x9csecretory peptidexe2x80x9d) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
A xe2x80x9csoluble receptorxe2x80x9d is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis. Soluble receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
The term xe2x80x9csplice variantxe2x80x9d is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as xe2x80x9caboutxe2x80x9d X or xe2x80x9capproximatelyxe2x80x9d X, the stated value of X will be understood to be accurate to xc2x110%.
All references cited herein are incorporated by reference in their entirety.
The present invention is based in part upon the discovery of a novel heterodimeric soluble receptor protein having the structure of a class I cytokine receptor. The heterodimeric soluble receptor includes at least one zalpha11 soluble receptor subunit, disclosed in the commonly owned U.S. patent application Ser. No. 09/404,641. A second soluble receptor polypeptide included in the heterodimeric soluble receptor belongs to the receptor subfamily that includes the IL-2 xcex3-common receptor (IL-2Rxcex3, or xcex3C), IL-2 receptor xcex2-subunit, and the xcex2-common receptor (i.e., IL3, IL-5, IL-13, IL-15 and GM-CSF receptor xcex2-subunits), IL-13xcex1, IL-13xcex1xe2x80x2, IL-15 receptor subunits, and the like. The soluble human and mouse zalpha11 receptor (IL-21R) monomer and homodimer was shown to antagonize of the activity of the natural ligand for the zalpha11 receptor, zalpha11 Ligand (IL-21) (Parrish-Novak, J. et al., Nature 408:57-63, 2000). The zalpha11 Ligand is disclosed in the commonly owned U.S. patent application Ser. No. 09/522,217. According to the present invention, a heterodimeric soluble zalpha11 receptor, as exemplified by a preferred embodiment of a soluble zalpha11 receptor+soluble IL-2Rxcex3 receptor heterodimer (zalpha11/IL-2Rxcex3), was shown to act as a potent antagonist of the zalpha11 Ligand. As disclosed in the examples herein, the preferred zalpha11/IL-2Rxcex3 heterodimer was a more effective antagonist zalpha11 Ligand activity, and hence more superior antagonist, than a zalpha11 homodimer or monomer.
Moreover, also contemplated by the present invention are homodimeric and monomeric zalpha11-comprising soluble receptors; as well as homodimeric, heterodimeric, and multimeric zalpha11-comprising receptors that are capable of intracellular signaling. Such receptors can comprise at least one an extracellular domain of a zaplha11 receptor, and an intracellular domain from zalpha11 or another class I cytokine receptor. The additional heterodimeric or multimeric subunit can comprise the extracellular domain from IL-2Rxcex3 receptor (e.g., SEQ ID NO:4), L-13xcex1 (also known as IL-13RA2; SEQ ID NO:84), IL-13xcex1xe2x80x2 (also known as IL-13RA1; SEQ ID NO:82, IL-15 (SEQ ID NO:86) receptor, or other class I receptor, and an intracellular domain from zalpha11 or another class I cytokine receptor.
The nucleotide sequence of a representative zalpha11-encoding DNA is described in SEQ ID NO:1 (from nucleotide 1 to 1614), and its deduced 538 amino acid sequence is described in SEQ ID NO:2. In its entirety, the zalpha11 polypeptide (SEQ ID NO:2) represents a full-length polypeptide segment (residue 1 (Met) to residue 538 (Ser) of SEQ ID NO:2). The domains and structural features of the zalpha11 polypeptide are further described below.
Analysis of the zalpha11 polypeptide encoded by the DNA sequence of SEQ ID NO:1 revealed an open reading frame encoding 538 amino acids (SEQ ID NO:2) comprising a predicted secretory signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Gly) of SEQ ID NO:2), and a mature polypeptide of 519 amino acids (residue 20 (Cys) to residue 538 (Ser) of SEQ ID NO:2). In addition to the WSXWS motif (SEQ ID NO:13) corresponding to residues 214 to 218 of SEQ ID NO:2, the receptor comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 20 (Cys) to 237 (His) of SEQ ID NO:2); a domain linker (residues 120 (Pro) to 123 (Pro) of SEQ ID NO:2); a penultimate strand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:2); a transmembrane domain (residues 238 (Leu) to 255 (Leu) of SEQ ID NO:2); complete intracellular signaling domain (residues 256 (Lys) to 538 (Ser) of SEQ ID NO:2) which contains a xe2x80x9cBox Ixe2x80x9d, signaling site (residues 267 (Ile) to 273 (Pro) of SEQ ID NO:2), and a xe2x80x9cBox IIxe2x80x9d signaling site (residues 301 (Leu) to 304 (Gly) of SEQ ID NO:2). Moreover, there is a STAT3 binding site (YXXQ) located near the C-terminus from residues 519 (Tyr) to 522 (Gln) of SEQ ID NO:2. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. In addition to these domains, conserved receptor features in the encoded receptor include (as shown in SEQ ID NO:2) a conserved Trp residue at position 138, and a conserved Arg residue at position 201. Moreover the zalpha11 contains conserved Cys residues typical of class I cytokine receptors, shown in residues 25, 35, 65, and 81 of SEQ ID NO:2, and corresponding regions of SEQ ID NO:6 and SEQ ID NO:69 described below. The corresponding polynucleotides encoding the zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO:1. The human zalpha11 soluble receptor polypeptide, comprising residues 20 (Cys) to 237 (His) of SEQ ID NO:2, is shown in SEQ ID NO:6, and the corresponding polynucleotide sequence for the human zalpha11 soluble receptor polypeptide is shown in SEQ ID NO:5.
SEQ ID NO:3 is a polynucleotide sequence comprising a fragment of the human IL-2Rxcex3 receptor that encodes a soluble 232 amino acid soluble IL-2Rxcex3 receptor polypeptide (SEQ ID NO:4). Those skilled in the art will recognize that these domain boundaries for the IL-2Rxcex3 receptor extracellular domain are approximate, and other soluble IL-2Rxcex3 receptor polypeptides, such as those including an IL-2Rxcex3 receptor polypeptide secretory signal sequence or additional IL-2Rxcex3 receptor polypeptide amino acids in the extracellular domain, are encompassed within the scope of the present invention.
A variant form of the human zalpha11 polypeptide was identified (WIPO publication No. WO 00/27822 shown as SEQ ID NO:3 and SEQ ID NO:4 therein) and is shown in the DNA sequence of SEQ ID NO:64; and corresponding polypeptide sequence shown in (SEQ ID NO:65). This particular alternative zalpha11 receptor polypeptide contains 568 amino acids, and comprises a predicted secretory signal peptide of 20 amino acid residues (residue 1 (Met) to residue 20 (Gly) of SEQ ID NO:65), and a mature polypeptide of 548 amino acids (residue 21 (Met) to residue 568 (Ser) of SEQ ID NO:65). In addition to the WSXWS motif (SEQ ID NO:13) corresponding to residues 244 to 248 of SEQ ID NO:65, the receptor comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 21 (Met) to 267 (His) of SEQ ID NO:65); no domain linker; a penultimate strand region (residues 222 (Lys) to 232 (Ala) of SEQ ID NO:65); a transmembrane domain (residues 268 (Leu) to 285 (Leu) of SEQ ID NO:65); complete intracellular signaling domain (residues 286 (Lys) to 568 (Ser) of SEQ ID NO:65) which contains a xe2x80x9cBox Ixe2x80x9d signaling site (residues 297 (Ile) to 303 (Pro) of SEQ ID NO:65), and a xe2x80x9cBox IIxe2x80x9d signaling site (residues 331 (Leu) to 334 (Gly) of SEQ ID NO:65). Moreover, there is a STAT3 binding site (YXXQ) located near the C-terminus from residues 549 (Tyr) to 552 (Gln) of SEQ ID NO:65. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. In addition to these domains, conserved receptor features in the encoded receptor include (as shown in SEQ ID NO:65) a conserved Trp residue at position 168, and a conserved Arg residue at position 231. The corresponding polynucleotides encoding the zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO:64. This particular human zalpha11 soluble receptor variant polypeptide, comprising residues 21 (Met) to 267 (His) of SEQ ID NO:65 (SEQ ID NO:69) and the corresponding polynucleotide sequence for this particular human zalpha11 soluble receptor polypeptide is shown in SEQ ID NO:68. This variant form of the human zalpha11 receptor is included in the heterodimeric and multimeric zalpha11 receptor complexes of the present invention, disclosed herein.
In addition, other variant forms of zalpha11 receptor are contemplated by the present invention, wherein the extracellular domain of the variant form disclosed above (e.g., 21 (Met) to 267 (His) of SEQ ID NO:65, or SEQ ID NO:69) comprises a domain linker comprising the amino acids PAPP (SEQ ID NO:70) inserted between amino acid 161 (Ser) and 162 (Arg) of SEQ ID NO:65, or the corresponding region of SEQ ID NO:69. A preferred domain linker comprises a sequence of amino acids from preferably 4 to 14 amino acids long, most preferably 14 amino acids long, wherein aside from the PAPP (SEQ ID NO:70) motif sequence any amino acid may be present. For example, a representative linker-containing variant zalpha11 soluble receptor is shown in SEQ ID NO:71. Moreover, other variant zalpha11 sequences can include, in reference to SEQ ID NO:65 a Gly at position 162 rather than an Arg, or the same Arg to Gly substitution in the corresponding region of SEQ ID NO:69 or SEQ ID NO:71, or other variant of SEQ ID NO:65 or SEQ ID NO:69 containing a domain linker as described above. Corresponding DNA sequences that encode such variants can be readily determined by one of skill in the art upon using the information present in Table 1 and Table 2.
The zalpha11 Ligand is a xe2x80x9cshort-helixxe2x80x9d form secreted four-helical bundle cytokine. The zalpha11 Ligand polynucleotide sequence is shown in SEQ ID NO:9 and corresponding amino acid sequence shown in SEQ ID NO:10. The secretory signal sequence comprises amino acid residues 1 (Met) to 31 (Gly), and the mature polypeptide comprises amino acid residues 32 (Gln) to 162 (Ser) (as shown in SEQ ID NO:10). In general, cytokines are predicted to have a four-alpha helix structure, with helices A, C and D being most important in ligand-receptor interactions, and are more highly conserved among members of the family. Referring to the human zalpha11 Ligand amino acid sequence shown in SEQ ID NO:10, alignment of human zalpha11 Ligand, human IL-15, human IL-4, and human GM-CSF amino acid sequences it is predicted that zalpha11 Ligand helix A is defined by amino acid residues 41-56; helix B by amino acid residues 69-84; helix C by amino acid residues 92-105; and helix D by amino acid residues 135-148; as shown in SEQ ID NO:10. Structural analysis suggests that the A/B loop is long, the B/C loop is short and the C/D loop is parallel long. Conserved cysteine residues within zalpha11 Ligand correspond to amino acid residues 71, 78, 122 and 125 of SEQ ID NO:10. Consistent cysteine placement is further confirmation of the four-helical-bundle structure. Also highly conserved in the family comprising IL-15, IL-2, IL-4, GM-CSF and zalpha11 Ligand is the Glu-Phe-Leu sequence as shown in SEQ ID NO:10 at residues 136-138.
Further analysis of zalpha11 Ligand based on multiple alignments of known cytokines predicts that amino acid residues 44, 47 and 135 (as shown in SEQ ID NO:10) play an important role in zalpha11 Ligand binding to its cognate receptor. Based on comparison between sequences of human and murine zalpha11 Ligand well-conserved residues were found in the regions predicted to encode alpha helices A and D. The corresponding polynucleotides encoding the zalpha11 Ligand polypeptide regions, domains, motifs, residues and sequences described herein are as shown in SEQ ID NO:9. The murine zalpha11 Ligand is shown in SEQ ID NO:46, and corresponding polypeptide sequence shown in SEQ ID NO:47.
The activity of molecules of the present invention can be measured using a variety of assays that measure proliferation of and/or binding to cells expressing the zalpha11 receptor. Of particular interest are changes in zalpha11 Ligand-dependent cells. A suitable cell line was engineered to be zalpha11 Ligand-dependent that comprises an IL-3-dependent BaF3 cell line (Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986). Moreover, other suitable cell lines to be engineered to be zalpha11 Ligand-dependent include FDC-P1 (Hapel et al., Blood 64: 786-790, 1984); and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growth factor-dependent cell lines can be established according to published methods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexter et al., in Baum et al. Eds., Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980).
Zalpha11 Ligand stimulates proliferation, activation, differentiation and/or induction or inhibition of specialized cell function of cells involved homeostasis of hematopoiesis and immune function. In particular, zalpha11 Ligand polypeptides stimulate proliferation, activation, differentiation, induction or inhibition of specialized cell functions of cells of the hematopoietic lineages, including, but not limited to, T cells, B cells, NK cells, dendritic cells, monocytes, and macrophages, as well as epithelial cells. Proliferation and/or differentiation of hematopoietic cells can be measured in vitro using cultured cells or in vivo by administering zalpha11 Ligand to the appropriate animal model. Assays measuring cell proliferation or differentiation are well known in the art and described herein. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990, incorporated herein by reference), incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989, incorporated herein by reference), incorporation of 5-bromo-2xe2x80x2-deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporated herein by reference), and use of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833, 1988; all incorporated herein by reference). Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporated herein by reference). Conversely, these assays can be used in a competition to assess the antagonist or zalpha11 Ligand binding activity of the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptors of the present invention. Moreover, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptors of the present invention can be used as an antagonist or Ligand binding agent to modulate the immune system and hematopoietic activities of the zalpha11 Ligand.
Zalpha11 Ligand was isolated from tissue known to have important immunological function and which contain cells that play a role in the immune system. Zalpha11 Ligand is expressed in CD3+ selected, activated peripheral blood cells, and it has been shown that zalpha11 Ligand expression increases after T cell activation. Moreover, results of experiments described in commonly owned U.S. patent application Ser. No. 09/522,217, and the Examples section herein, demonstrate that zalpha11 Ligand has an effect on the growth/expansion and/or differentiated state of NK cells or NK progenitors. Additional evidence demonstrates that zalpha11 Ligand affects proliferation and/or differentiation of T cells and B cells in vivo. Factors that both stimulate proliferation of hematopoietic progenitors and activate mature cells are generally known. NK cells are responsive to IL-2 alone, but proliferation and activation generally require additional growth factors. For example, it has been shown that IL-7 and Steel Factor (c-kit ligand) were required for colony formation of NK progenitors. IL-15+IL-2 in combination with IL-7 and Steel Factor was more effective (Mrxc3x3zek et al., Blood 87:2632-2640, 1996). However, unidentified cytokines may be necessary for proliferation of specific subsets of NK cells and/or NK progenitors (Robertson et. al., Blood 76:2451-2438, 1990). A composition comprising zalpha11 Ligand and IL-15 stimulates NK progenitors and NK cells, with evidence that this composition is more potent than previously described factors and combinations of factors. Such compositions can further comprise kit ligand or stem cell factor. Thus, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptors of the present invention can be used as an antagonist or Ligand binding agent to decrease the activity of the zalpha11 Ligand on NK cells.
Moreover, the tissue distribution of a receptor for a given cytokine offers a strong indication of the potential sites of action of that cytokine. Northern analysis of zalpha11 receptor revealed transcripts in human spleen, thymus, lymph node, bone marrow, and peripheral blood leukocytes. Specific cell types were identified as expressing zalpha11 receptors, and strong signals were seen in a mixed lymphocyte reaction (MLR) and in the Burkitt""s lymphoma Raji. The two monocytic cell lines, THP-1 (Tsuchiya et al., Int. J. Cancer 26:171-176, 1980) and U937 (Sundstrom et al., Int. J. Cancer 17:565-577, 1976), were negative. Zalpha11 receptor is expressed at relatively high levels in the MLR, in which peripheral blood mononuclear cells (PBMNC) from two individuals are mixed, resulting in mutual activation. Detection of high levels of transcript in the MLR but not in resting T or B cell populations suggests that zalpha11 receptor expression may be induced in one or more cell types during activation. Activation of isolated populations of T and B cells can be artificially achieved by stimulating cells with PMA and Ionomycin. When sorted cells were subjected to these activation conditions, levels of zalpha11 receptor transcript increased in both cell types, supporting a role for this receptor and zalpha11 Ligand in immune responses, especially in autocrine and paracrine T and B cell expansions during activation. Zalpha11 Ligand may also play a role in the expansion of more primitive progenitors involved in lymphopoiesis. Thus, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptors of the present invention can be used as an antagonist or Ligand binding agent to modulate the lymphopoietic activities of the zalpha11 Ligand.
Zalpha11 receptor was found to be present at low levels in resting T and B cells, and was upregulated during activation in both cell types. Interestingly, the B cells also down-regulate the message more quickly than do T cells, suggesting that amplitude of signal and timing of quenching of signal are important for the appropriate regulation of B cell responses.
In addition, a large proportion of intestinal lamina propria cells show positive hybridization signals with zalpha11 receptor. This tissue consists of a mixed population of lymphoid cells, including activated CD4+ T cells and activated B cells. Immune dysfunction, in particular chronic activation of the mucosal immune response, plays an important role in the etiology of Crohn""s disease and inflammatory bowel disease (IBD); abnormal response to and/or production of proinflammatory cytokines is also a suspected factor (Braegger et al., Annals Allergy 72:135-141, 1994; Sartor RB Am. J. Gastroenterol. 92:5S-11S, 1997). The zalpha11 Ligand in concert with IL-15 expands NK cells from bone marrow progenitors and augments NK cell effector function. Zalpha11 Ligand also co-stimulates mature B cells stimulated with anti-CD40 antibodies, but inhibits B cell proliferation to signals through IgM. Zalpha11 Ligand enhances T cell proliferation in concert with a signal through the T cell receptor, and over expression in transgenic mice leads to lymphopenia and an expansion of monocytes and granulocytes. These pleiotropic effects of zalpha11 Ligand suggest that molecules that antagonize or bind zalpha11 Ligand, such as the molecules of the present invention, can provide therapeutic utility for a wide range of diseases arising from defects in the immune system, including (but not limited to) systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, Crohn""s Disease, IBD, and diabetes. It is important to note that these diseases are the result of a complex network of immune dysfunction (SLE, for example, is the manifestation of defects in both T and B cells), and that immune cells are dependent upon interaction with one another to elicit a potent immune response. Therefore, zalpha11 Ligand (or an antagonist of the Ligand, such a molecule of the present invention) that can be used to manipulate more than one type of immune cell is an attractive therapeutic candidate for intervention at multiple stages of disease.
Similarly, the tissue distribution of the mRNA corresponding to IL-2Rxcex3 receptor cDNA shows expression in hematopoietic and lymphoid cells including CD4+ T-cells, CD8+ T-cells, CD20+ B-cells, CD56+ NK cells, CD 14+ monocytes, as well as granulocytes. IL-2Rxcex3 receptor cDNA is generally not found in other cell types, including epithelial cells and fibroblast cells. The expression pattern of this receptor correlates with the activities of the zalpha11 Ligand and the localization of the zalpha11 receptor. Moreover, antibodies to the IL-2Rxcex3 receptor decrease or ablate the effect of zalpha11 Ligand in B-cells and BaF3/zalpha11 receptor cells, demonstrating that the zalpha11 receptor and IL-2Rxcex3 receptor can heterodimerize in vivo and in vitro.
The zalpha11 Ligand both promotes expansion of NK cell populations from bone marrow and regulates the proliferation of mature T and B cells in response to activating stimuli. The zalpha11 Ligand acts through a receptor complex that includes at least one zalpha11 receptor subunit and the xcex3C subunit of IL2R, even though the cytoplasmic domain of zalpha11 receptor is capable of transducing signal in a homodimeric configuration (commonly owned U.S. patent application Ser. No. 09/522,217). IL4Rxcex1 is also capable of signaling as a homodimer (Kammer, W. et al., J. Biol. Chem. 271:23634-23637, 1996), although the true functional IL4 receptor complex is a IL4Rxcex1/xcex3C heterodimer. Signaling in BaF3/zalpha11 receptor could have resulted from interactions of the human zalpha11 receptor with endogenous murine xcex3C, and Examples herein show that antibodies to the xcex3C subunit decrease zalpha11 Ligand signaling in these cells.
Moreover, the IL2 receptor has been studied in detail and is composed of an xcex1-xcex2-xcex3C heterotrimer. The xcex2 and xcex3C subunits are both essential for signal transduction and are members of the hematopoietin receptor superfamily (Cosman, D., Cytokine 5:95-106, 1993), whereas the xcex1 subunit appears to primarily be involved in high-affinity binding conversion and is structurally distinct from the hematopoietin receptor family. The xcex3C subunit has been shown to participate in forming the receptors for IL4, IL7, IL9, and IL15, in addition to IL2 (for review, see Sugamura, K., et al., Annu. Rev. Immunol. 14:179-205, 1996), and null mutations in the xcex3C gene have been shown to cause X-linked severe combined immunodeficiency (X-SCID) (Noguchi, M. et al., Cell 73:147-157, 1993).
Zalpha11 Ligand antagonism of anti-IgM and IL4-induced B cell proliferation (commomly owned U.S. patent application Ser. No. 09/522,217, and examples herein) could be due to competition for xcex3C; however, it is clear that IL4 can signal through a xcex3C-independent receptor (IL4Rxcex1+IL13Rxcex1xe2x80x2) (Murata, T. et al., Blood 91:3884-3891, 1998). B cells from human SCID patients proliferate normally in response to anti-IgM and IL4 (Matthews, D. J. et al., Blood 85:38-42, 1995), and IL4 responsiveness in normal human B cells is associated with both IL13 responsiveness and levels of IL13R (Ford, D. et al., J. Immunol. 163:3185-3193, 1999). Similarly, Zalpha11 Ligand may signal through a heterodimeric, heterotrimeric or multimeric complex that includes zalpha11 receptor and a non-xcex3C-subunit. As such, the present invention contemplates soluble zalpha11 receptor heterodimeric antagonists and binding agents to the zalpha11 Ligand that do not include the xcex3C subunit, but include an additional Class I cytokine subunit, for example, IL13Rxcex1xe2x80x2 and the like.
The soluble receptors of the present invention are useful as antagonists of the zalpha11 Ligand cytokine. Such antagonistic effects can be achieved by direct neutralization or binding of the zalpha11 Ligand. In addition to antagonistic uses, the soluble receptors of the present invention can bind zalpha11 Ligand and act as carrier proteins for the zalpha11 Ligand cytokine, in order to transport the Ligand to different tissues, organs, and cells within the body. As such, the soluble receptors of the present invention can be fused or coupled to molecules, polypeptides or chemical moieties that direct the soluble-receptor-Ligand complex to a specific site, such as a tissue, specific immune cell, or tumor. Thus, the soluble receptors of the present invention can be used to specifically direct the action of the zalpha11 Ligand. See, Cosman, D. Cytokine 5: 95-106, 1993; and Femandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.
Moreover, the soluble receptors of the present invention can be used to stabilize the zalpha11 Ligand, to increase the bio-availability, therapeutic longevity, and/or efficacy of the Ligand by stabilizing the Ligand from degradation or clearance, or by targeting the ligand to a site of action within the body. For example the naturally occurring IL-6/soluble IL-6R complex stabilizes IL-6 and can signal through the gp130 receptor. See, Cosman, D. supra., and Femandez-Botran, R. supra.
For example, the Zalpha11 Ligand will be useful in treating tumorgenesis, and therefore would be useful in the treatment of cancer. Zalpha11 Ligand inhibits IL-4 stimulated proliferation of anti-IgM stimulated normal B-cells and a similar effect is observed in B-cell tumor lines suggesting that there may be therapeutic benefit in treating patients with the zalpha11 Ligand in order to induce the B cell tumor cells into a less proliferative state. The ligand could be administered in combination with other agents already in use including both conventional chemotherapeutic agents as well as immune modulators such as interferon alpha. Alpha/beta interferons have been shown to be effective in treating some leukemias and animal disease models, and the growth inhibitory effects of interferon-alpha and zalpha11 Ligand are additive for at least one B-cell tumor-derived cell line. Moreover, stabilization of the zalpha11 Ligand or ability to target the Ligand to specific sites of action with the soluble receptors of the present invention would be desirable in this therapeutic endeavor.
The present invention provides a method of reducing proliferation of neoplastic B or T cells comprising administering to a mammal with a B or T cell neoplasm an amount of a composition of zalpha11 Ligand antagonist, such as the soluble receptors of the present invention, sufficient to reduce proliferation of the neoplastic B or T cells. In other embodiments, the composition can comprise at least one other cytokine selected from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand or stem cell factor. Furthermore, the zalpha11 Ligand antagonist can be a toxic fusion. Similarly, soluble receptor-toxic fusions and soluble receptors of the present invention can be used to reduce proliferation of lymphoid and hematopoietic neoplasms that over-express or grow in response to zalpha11 Ligand. Moreover, indirect effects of the soluble receptors of the present invention can modulate NK cell function induced by zalpha11 Ligand, and hence indirectly enhance tumor cell killing.
The present invention provides polynucleotide molecules, including DNA and RNA molecules that encode the heterodimeric zalpha11 receptor polypeptides disclosed herein. Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:7 is a degenerate DNA sequence that encompasses all DNAs that encode the soluble zalpha11 receptor polypeptide of SEQ ID NO:6. SEQ ID NO:66 is a degenerate DNA sequence that encompasses all DNAs that encode the soluble zalpha11 receptor polypeptide of SEQ ID NO:69. SEQ ID NO:8 is a degenerate DNA sequence that encompasses all DNAs that encode the soluble human IL-2Rxcex3 polypeptide of SEQ ID NO:4. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:7, SEQ ID NO:66 and SEQ ID NO:8 also provide all RNA sequences encoding SEQ ID NO:6, SEQ ID NO:69 and SEQ ID NO:4 respectively by substituting U for T. Thus, zalpha11 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 654 of SEQ ID NO:7 or comprising nucleotide 1 to nucleotide 741 of SEQ ID NO:66, soluble human IL-2Rxcex3 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 696 of SEQ ID NO:8, and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO:7, SEQ ID NO:66 and SEQ ID NO:8 to denote degenerate nucleotide positions. xe2x80x9cResolutionsxe2x80x9d are the nucleotides denoted by a code letter. xe2x80x9cComplementxe2x80x9d indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
The degenerate codons used in SEQ ID NO:7, SEQ ID NO:66 and SEQ ID NO:8 encompass all possible codons for a given amino acid, are set forth in Table 2.
One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:6, SEQ ID NO:69 or SEQ ID NO:4. Variant sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species can exhibit xe2x80x9cpreferential codon usage.xe2x80x9d In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term xe2x80x9cpreferential codon usagexe2x80x9d or xe2x80x9cpreferential codonsxe2x80x9d is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO:7, SEQ ID NO:66 and SEQ ID NO:8 serves as a template for optimizing expression of polynucleotides and polypeptides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:5, SEQ ID NO:68 or SEQ ID NO:3, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Numerous equations for calculating Tm are known in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto, Calif.), as well as sites on the Internet, are available tools for analyzing a given sequence and calculating Tm based on user-defined criteria. Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences (e.g.,  greater than 50 base pairs) is performed at temperatures of about 20-25xc2x0 C. below the calculated Tm. For smaller probes (e.g.,  less than 50 base pairs) hybridization is typically carried out at the Tm or 5-10xc2x0 C. below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the Tm of the hybrid about 1xc2x0 C. for each 1% formamide in the buffer solution. Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42xc2x0 C. in a solution comprising: about 40-50% formamide, up to about 6xc3x97SSC, about 5xc3x97Denhardt""s solution, zero up to about 10% dextran sulfate, and about 10-20 xcexcg/ml denatured commercially-available carrier DNA. Generally, such stringent conditions include temperatures of 20-70xc2x0 C. and a hybridization buffer containing up to 6xc3x97SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2xc3x97SSC. For example, a suitable wash stringency is equivalent to 0.1xc3x97SSC to 2xc3x97SSC, 0.1% SDS, at 55xc2x0 C. to 65xc2x0 C. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes. Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zalpha11 receptor RNA or the RNA for the heterodimeric component of the receptor, such as IL-2Rxcex3, or other class I cytokine receptor. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include PBLs, spleen, thymus, and lymph tissues, Raji cells, human erythroleukemia cell lines (e.g., TF-1), acute monocytic leukemia cell lines, other lymphoid and hematopoietic cell lines, and the like, for the zalpha11 receptor. RNA for a heterodimeric component of the receptor, such as IL-2Rxcex3, or other class I cytokine receptor can be isolated from lymphoid cells, such as those described above, and other cells and tissues as is known in the art for these receptors. Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)+ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)+ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zalpha11 polypeptides are then identified and isolated by, for example, hybridization or polymerase chain reaction (PCR) (Mullis, U.S. Pat. No. 4,683,202).
The polynucleotides of the present invention can also be synthesized using DNA synthesis machines. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short polynucleotides (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. However, for producing longer polynucleotides ( greater than 300 bp), special strategies are usually employed, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
An alternative way to prepare a full-length gene is to synthesize a specified set of overlapping oligonucleotides (40 to 100 nucleotides). After the 3xe2x80x2 and 5xe2x80x2 short overlapping complementary regions (6 to 10 nucleotides) are annealed, large gaps still remain, but the short base-paired regions are both long enough and stable enough to hold the structure together. The gaps are filled and the DNA duplex is completed via enzymatic DNA synthesis by E. coli DNA polymerase I. After the enzymatic synthesis is completed, the nicks are sealed with T4 DNA ligase. Double-stranded constructs are sequentially linked to one another to form the entire gene sequence which is verified by DNA sequence analysis. See Glick and Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. Moreover, other sequences are generally added that contain signals for proper initiation and termination of transcription and translation.
The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are heterodimeric soluble receptor complexes combining soluble zalpha11 receptor and soluble human IL-2Rxcex3 or other soluble Class I cytokine receptor polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Known and unknown orthologs of human soluble zalpha11 receptor and soluble human IL-2Rxcex3 or other soluble Class I cytokine receptors can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type, such as lymphoid cells, that expresses zalpha11receptor, human IL-2Rxcex3 or other Class I cytokine receptors. Moreover, suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zalpha11-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using PCR (Mullis, supra.), using primers designed from the representative human zalpha11 sequence, or soluble human IL-2Rxcex3 sequence, disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zalpha11 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
Cytokine receptor subunits are characterized by a multi-domain structure comprising an extracellular domain, a transmembrane domain that anchors the polypeptide in the cell membrane, and an intracellular domain. The extracellular domain the zalpha11 receptor is a ligand-binding domain, that binds zalpha11 Ligand, and the intracellular domain is an effector domain involved in signal transduction, although ligand-binding and effector functions can reside on separate subunits of a multimeric receptor. The ligand-binding domain may itself be a multi-domain structure. Multimeric receptors include homodimers (e.g., PDGF receptor xcex1xcex1 and xcex2xcex2 isoforms, erythropoietin receptor, MPL, and G-CSF receptor), heterodimers whose subunits each have ligand-binding and effector domains (e.g., PDGF receptor xcex1xcex2 isoform), and multimers having component subunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-13, IL-15 and GM-CSF receptors). Some receptor subunits are common to a plurality of receptors. For example, the AIC2B subunit, which cannot bind ligand on its own but includes an intracellular signal transduction domain, is a component of IL-3 and GM-CSF receptors. Many cytokine receptors can be placed into one of four related families on the basis of the structure and function. Hematopoietic receptors, for example, are characterized by the presence of a domain containing conserved cysteine residues and the WSXWS motif (SEQ ID NO:13). Cytokine receptor structure has been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991; and Cosman, Cytokine 5:95-106, 1993. Under selective pressure for organisms to acquire new biological functions, new receptor family members likely arise from duplication of existing receptor genes leading to the existence of multi-gene families. Family members thus contain vestiges of the ancestral gene, and these characteristic features can be exploited in the isolation and identification of additional family members. Thus, the cytokine receptor superfamily is subdivided into several families, for example, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGF receptors); the hematopoietin family (including IL-2 receptor xcex2-subunit, GM-CSF receptor xcex1-subunit, GM-CSF receptor xcex2-subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13 and IL-15 receptors); TNF receptor family (including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGF receptor).
Analysis of the zalpha11 receptor sequence suggests that it is a member of the same receptor subfamily as the IL-2 receptor xcex2-subunit, IL-4, and IL-9, receptors. Certain receptors in this subfamily (e.g., EPO-R or MPL) associate to form homodimers that transduce a signal. Other members of the subfamily (e.g., IL-6, IL-11, and LIF receptors) combine with a second subunit (termed a xcex2-subunit) to bind ligand and transduce a signal. Specific xcex2-subunits associate with a plurality of specific cytokine receptor subunits. For example, the xcex2-subunit gp130 (Hibi et al., Cell 63:1149-1157, 1990) associates with receptor subunits specific for IL-6, IL-11, and LIF (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755). Oncostatin M binds to a heterodimer of LIF receptor and gp130. CNTF binds to trimeric receptors comprising CNTF receptor, LIF receptor, and gp130 subunits. Moreover, IL-4 and IL-13 elicit responses through the IL-4 and IL-13 receptors by acting upon various functional heterodimeric receptor complexes, e.g. with and without the xcex3C subunit, and such heterodimeric receptor complexes may affect whether the cytokines act upon hematopoietic or non-hematopoietic cells (Andersson, A. et al., Eur. J. Immunol. 27:1762-1768, 1997; Murata, T. et al., Blood 10:3884-3891, 1998). Moreover, binding affinity of IL-4 on its receptor is increased when the xcex3C subunit of the IL4R complex is replaced by an IL-13Rxcex1xe2x80x2 subunit (Murata, T. et al., supra.). Thus, the soluble receptors of the present invention include zalpha11 receptor homodimers; and heterodimers that have a zalpha11 receptor component, such as soluble zalpha11/IL-2Rxcex3 or soluble zalpha11 receptor heterodimerized with another soluble Class I cytokine receptor, such as IL-13Rxcex1 (SEQ ID NO:84), IL-13Rxcex1xe2x80x2 (SEQ ID NO:82) or an IL-15 (SEQ ID NO:86) receptor subunit.
For example, suitable Class I cytokine soluble receptors that can heterodimerize with a soluble zalpha11 receptor component (e.g., SEQ ID NO:6), include a soluble receptor for IL-13Rxcex1 as shown in SEQ ID NO:84, IL-13Rxcex1xe2x80x2 as shown in SEQ ID NO:82, or IL-15 as shown in SEQ ID NO:86. Morevoer, functional sub-fragments, such as minimal cytokine binding fragments, of these Class I cytokine soluble receptors can be used. Such functional fragments include 1 to 322, 7 to 322, and 105 to 322 of SEQ ID NO:82; 1 to 317, 10 to 317, and 105 to 317 of SEQ ID NO:84; and 1 to 173 of SEQ ID NO:86. The corresponding polynucleotide sequences are as shown in SEQ ID NO:81, SEQ ID NO:83 and SEQ ID NO:85 respecitvels. It is well within the level of one of skill in the art to delineate what sequences of a known class I cytokine sequence comprise the extracellular cytokine binding domain free of a transmembrane domain and intracellular domain.
A polynucleotide sequence for the mouse ortholog of human zalpha11 receptor has been identified and is shown in SEQ ID NO:11 and the corresponding amino acid sequence shown in SEQ ID NO:12. Analysis of the mouse zalpha11 polypeptide encoded by the DNA sequence of SEQ ID NO:11 revealed an open reading frame encoding 529 amino acids (SEQ ID NO:12) comprising a predicted secretory signal peptide of 19 amino acid residues (residue 1 (Met) to residue 19 (Ser) of SEQ ID NO:12), and a mature polypeptide of 510 amino acids (residue 20 (Cys) to residue 529 (Ser) of SEQ ID NO:2). In addition to the WSXWS motif (SEQ ID NO:13) corresponding to residues 214 to 218 of SEQ ID NO:12, the receptor comprises a cytokine-binding domain of approximately 200 amino acid residues (residues 20 (Cys) to 237 (His) of SEQ ID NO:12); a domain linker (residues 120 (Pro) to 123 (Pro) of SEQ ID NO:12); a penultimate strand region (residues 192 (Lys) to 202 (Ala) of SEQ ID NO:12); a transmembrane domain (residues 238 (Met) to 254 (Leu) of SEQ ID NO:12); complete intracellular signaling domain (residues 255 (Lys) to 529 (Ser) of SEQ ID NO:12) which contains a xe2x80x9cBox Ixe2x80x9d signaling site (residues 266 (Ile) to 273 (Pro) of SEQ ID NO:12), and a xe2x80x9cBox IIxe2x80x9d signaling site (residues 301 (Ile) to 304 (Val) of SEQ ID NO:2). A comparison of the human and mouse amino acid sequences reveals that both the human and orthologous polypeptides contain corresponding structural features described above. The mature sequence for the mouse zalpha11 begins at Cys20 (as shown in SEQ ID NO:12), which corresponds to Cys20 (as shown in SEQ ID NO:2) in the human sequence. There is about 69% identity a between the mouse and human zalpha11 sequences over the extracellular cytokine binding domain corresponding to residues 20 (Cys) to 237 (His) of SEQ ID NO:2 (SEQ ID NO:6) and residues 20 (Cys) to 237 (His) of SEQ ID NO:12. The above percent identities were determined using a FASTA program with ktup=1, gap opening penalty=12, gap extension penalty=2, and substitution matrix=BLOSUM62, with other FASTA program parameters set as default. The corresponding polynucleotides encoding the mouse zalpha11 polypeptide regions, domains, motifs, residues and sequences described above are as shown in SEQ ID NO:11.
The present invention also provides for a heterodimeric soluble receptor, wherein the isolated soluble zalpha11 receptor polypeptide therein is substantially similar to the polypeptides of SEQ ID NO:6 and their orthologs. Moreover, in a preferred embodiment, the present invention also provides for a heterodimeric soluble receptor, wherein an isolated soluble IL-2Rxcex3 receptor polypeptide therein is substantially similar to the polypeptides of SEQ ID NO:4 and their orthologs. The term xe2x80x9csubstantially similarxe2x80x9d is used herein to denote polypeptides having at least 70%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:6 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:6 or SEQ ID NO:4 their orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the xe2x80x9cblosum 62xe2x80x9d scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:             Total      ⁢              xe2x80x83            ⁢      number      ⁢              xe2x80x83            ⁢      of      ⁢              xe2x80x83            ⁢      identical      ⁢              xe2x80x83            ⁢      matches                                            [                          length              ⁢                              xe2x80x83                            ⁢              of              ⁢                              xe2x80x83                            ⁢              the              ⁢                              xe2x80x83                            ⁢              longer              ⁢                              xe2x80x83                            ⁢              sequence              ⁢                              xe2x80x83                            ⁢              plus              ⁢                              xe2x80x83                            ⁢              the                        ⁢                          xe2x80x83                                                                        number            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            gaps            ⁢                          xe2x80x83                        ⁢            introduced            ⁢                          xe2x80x83                        ⁢            into            ⁢                          xe2x80x83                        ⁢            the            ⁢                          xe2x80x83                        ⁢            longer                                                                          sequence              ⁢                              xe2x80x83                            ⁢              in              ⁢                              xe2x80x83                            ⁢              order              ⁢                              xe2x80x83                            ⁢              to              ⁢                              xe2x80x83                            ⁢              align              ⁢                              xe2x80x83                            ⁢              the              ⁢                              xe2x80x83                            ⁢              two              ⁢                              xe2x80x83                            ⁢              sequences                        ]                                xc3x97  100
Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above.
Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The xe2x80x9cFASTAxe2x80x9d similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zsig57. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat""l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990).
Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:6) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are xe2x80x9ctrimmedxe2x80x9d to include only those residues that contribute to the highest score. If there are several regions with scores greater than the xe2x80x9ccutoffxe2x80x9d value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred program parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution matrix=BLOSUM62, with other parameters set as default. These FASTA program parameters can be introduced into a FASTA program by modifying the scoring matrix file (xe2x80x9cSMATRIXxe2x80x9d), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other program parameters set as default.
The BLOSUM62 table (Table 3) is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat""l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed below), the language xe2x80x9cconservative amino acid substitutionxe2x80x9d preferably refers to a substitution represented by a BLOSUM62 value of greater than xe2x88x921. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
Variant zalpha11 polypeptides or substantially homologous zalpha11 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes zalpha11 soluble receptor polypeptides of from about 190 to about 245 amino acid residues that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO:6. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zalpha11 polypeptide and the affinity tag. Suitable sites include thrombin cleavage sites and factor Xa cleavage sites.
The present invention further provides a variety of other polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains, e.g., IgGxcex31, and the human xcexa light chain. Inmunoglobulin-soluble zalpha11 receptor or immunoglobulin-soluble zalpha11 heterodimeric polypeptide, such as immunoglobulin-soluble zalpha11/IL-2Rxcex3 fusions can be expressed in genetically engineered cells to produce a variety of multimeric zalpha11 receptor analogs. Auxiliary domains can be fused to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 to target them to specific cells, tissues, or macromolecules (e.g., collagen, or cells expressing the zalpha11 Ligand). A zalpha11 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zalpha11 amino acid residues.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. ligand binding and signal transduction) as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites of ligand-receptor, protein-protein or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related receptors.
Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined. Within these regions one can determine specific residues that will be more or less tolerant of change and maintain the overall tertiary structure of the molecule. Methods for analyzing sequence structure include, but are not limited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight II(copyright) viewer and homology modeling tools; MSI, San Diego, Calif.), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995; and, Cordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general, when designing modifications to molecules or identifying specific fragments determination of structure will be accompanied by evaluating activity of modified molecules.
Amino acid sequence changes are made in soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, so as to minimize disruption of higher order structure essential to biological activity. For example, where the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL2Rxcex3 comprises one or more helices, changes in amino acid residues will be made so as not to disrupt the helix geometry and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to the zalpha11 Ligand, or antagonizing zalpha11 Ligand activity. The effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed above or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are well known in the art compare folding of a variant protein to a standard molecule (e.g., the native protein). For example, comparison of the cysteine pattern in a variant and standard molecules can be made. Mass spectrometry and chemical modification using reduction and alkylation provide methods for determining cysteine residues which are associated with disulfide bonds or are free of such associations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem. 66:3727-3732, 1994). It is generally believed that if a modified molecule does not have the same disulfide bonding pattern as the standard molecule folding would be affected. Another well known and accepted method for measuring folding is circular dichrosism (CD). Measuring and comparing the CD spectra generated by a modified molecule and standard molecule is routine (Johnson, Proteins 7:205-214, 1990). Crystallography is another well known method for analyzing folding and structure. Nuclear magnetic resonance (NMR), digestive peptide mapping and epitope mapping are also known methods for analyzing folding and structural similarities between proteins and polypeptides (Schaanan et al., Science 257:961-964, 1992).
A Hopp/Woods hydrophilicity profile of the zalpha11 protein sequence as shown in SEQ ID NO:6 can be generated (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169, 1998). The profile is based on a sliding six-residue window. Buried G, S, and T residues and exposed H, Y, and W residues were ignored. For example, in the soluble zalpha11 receptor, hydrophilic regions include amino acid residues 55 through 60 of SEQ ID NO:2, amino acid residues 56 through 61 of SEQ ID NO: 2, amino acid residues 139 through 144 of SEQ ID NO: 2, and amino acid residues 227 through 232 of SEQ ID NO: 2. The corresponding hydrophilic regions in reference to SEQ ID NO:6 can be made with cross-reference to the above amino acid residues of SEQ ID NO:2.
Those skilled in the art will recognize that hydrophilicity or hydrophobicity will be taken into account when designing modifications in the amino acid sequence of a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, so as not to disrupt the overall structural and biological profile. Of particular interest for replacement are hydrophobic residues selected from the group consisting of Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant of substitution could include such residues as shown in SEQ ID NO:6, SEQ ID NO:69 and SEQ ID NO:4. However, Cysteine residues could be relatively intolerant of substitution.
The identities of essential amino acids can also be inferred from analysis of sequence similarity between Class I cytokine receptor family members with soluble zalpha11 receptor or soluble IL-2Rxcex3 receptor. Using methods such as xe2x80x9cFASTAxe2x80x9d analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions. An alternative approach to identifying a variant extracellular domain zalpha11 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zalpha11 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:5, or SEQ ID NO:68 as discussed above. Likewise, variants of soluble class I cytokine receptor contained within a zalpha11 heterodimeric polypeptide, such as the soluble IL-2Rxcex3 receptor component in soluble zalpha11/IL-2Rxcex3, can be identified as described above.
Other methods of identifying essential amino acids in the polypeptides of the present invention are procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci. USA 88:4498 (1991), Coombs and Corey, xe2x80x9cSite-Directed Mutagenesis and Protein Engineering,xe2x80x9d in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699 (1996).
The present invention also includes functional fragments of soluble zalpha11 receptor polypeptides or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides and nucleic acid molecules encoding such functional fragments. A xe2x80x9cfunctionalxe2x80x9d soluble zalpha11 receptor polypeptide or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide, or fragment thereof defined herein is characterized by its by its ability to bind to an anti-zalpha11 antibody, or to zalpha11 Ligand (either soluble or immobilized), or to antagonize zalpha11 Ligand activity in, for example, a biological or binding assay. As previously described herein, zalpha11 receptor is characterized by a class I cytokine receptor structure. Thus, the present invention further provides fusion proteins encompassing: (a) homodimeric or multimeric polypeptide molecules comprising an extracellular domain described herein; and (b) functional fragments comprising one or more of these domains. The other polypeptide portion of the fusion protein may be contributed by another class I cytokine receptor, for example, IL-2Rxcex3, IL-2 receptor xcex2-subunit and the xcex2-common receptor (i.e., IL3, IL-5, and GM-CSF receptor xcex2-subunits), IL-13xcex1, IL-13xcex1xe2x80x2, or IL-15 receptor subunits, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the soluble fusion protein.
Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encode a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3. As an illustration, DNA molecules having the nucleotide sequence of SEQ ID NO:1 or fragments thereof, can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for antagonizing zalpha11 Ligand biological or zalpha11 Ligand binding activity; or for the ability to bind anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide antibodies; or for the ability to bind zalpha11 Ligand. One alternative to exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired polypeptide fragment. Alternatively, particular fragments of a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide polynucleotide can be synthesized using the polymerase chain reaction.
Standard methods for identifying functional domains, such as Ligand binding domains, are routine for those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques for functional analysis of proteins are described by, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993); Content et al., xe2x80x9cExpression and preliminary deletion analysis of the 42 kDa 2-5A synthetase induced by human interferon,xe2x80x9d in Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, xe2x80x9cThe EGF Receptor,xe2x80x9d in Control of Animal Cell Proliferation 1, Boynton et al., (eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al., Plant Molec. Biol. 30:1 (1996).
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/062045) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Variants of the disclosed soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid xe2x80x9cevolutionxe2x80x9d of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect zalpha11 Ligand antagonist or binding activity in host cells of cloned, mutagenized soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below and in the Examples. Mutagenized DNA molecules that encode active receptors or portions thereof (e.g., ligand-binding fragments, and the like) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
The present invention thus provides a series of novel, hybrid molecules in which a segment comprising one or more of the domains of soluble zalpha11 receptor is fused to another soluble receptor polypeptide. Fusion is preferably done by splicing at the DNA level to allow expression of chimeric molecules in recombinant production systems. The resultant molecules are then assayed for such properties as improved solubility, improved stability, prolonged clearance half-life, improved expression and secretion levels, and pharmicodynamics. Such hybrid molecules may further comprise additional amino acid residues (e.g. a polypeptide linker) between the component proteins or polypeptides.
Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO:6 that retain zlpha11 Ligand binding or antagonist activity. For example, one can make a zalpha11 soluble receptor by preparing a variety of polypeptides that are substantially homologous to the cytokine-binding domain (residues 20 (Cys) to 237 (His) of SEQ ID NO:2 (SEQ ID NO:6), or a subsequence therein that binds zalpha11 Ligand, or allelic variants or species orthologs thereof) and retain ligand-binding activity of the wild-type zalpha11 protein. Such polypeptides may include additional amino acids from, for example, part or all of the signal peptide sequence, transmembrane and intracellular domains. Such polypeptides may also include additional polypeptide segments as generally disclosed herein such as labels, affinity tags, and the like. Similarly, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO:4, or other soluble class I cytokine receptors that form heterodimers with zalpha11 receptor.
For any soluble zalpha11 receptor polypeptide, including variants, and fusion polypeptides or proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
The soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides of the present invention, including full-length soluble receptor polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zalpha11 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
To direct a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zalpha11 receptor disclosed herein, the IL-2Rxcex3 (amino acid 1 (Met) to 22 (Gly) of SEQ ID NO:18), or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the zalpha11 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5xe2x80x2 to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus (CMV). See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as xe2x80x9ctransfectantsxe2x80x9d. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as xe2x80x9cstable transfectants.xe2x80x9d A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as xe2x80x9camplification.xe2x80x9d Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting, flow cytometry, or magnetic bead separation technology.
Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman and Hall; O""Reilly, D. R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, N J, Humana Press, 1995. A second method of making recombinant zalpha11 baculovirus utilizes a transposon-based system described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79, 1993). This system, which utilizes transfer vectors, is sold in the Bac-to-Bac(trademark) (Life Technologies, Rockville, Md.). This system utilizes a transfer vector, pFastBac1(trademark) (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zalpha11 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a xe2x80x9cbacmid.xe2x80x9d See, Hill-Perkins, M. S. and Possee, R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem 270:1543-9, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zalpha11 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique known in the art, a transfer vector containing zalpha11 is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zalpha11 is subsequently produced. Recombinant viral stocks are made by methods commonly used in the art.
The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO(trademark) cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II(trademark) (Life Technologies) or ESF 921(trademark) (Expression Systems) for the Sf9 cells; and Ex-cellO405(trademark) (JRH Biosciences, Lenexa, Kans.) or Express FiveO(trademark) (Life Technologies) for the T. ni cells. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R. D., ibid.; O""Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequent purification of the zalpha11 polypeptide from the supernatant can be achieved using methods described herein.
Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 5 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533.
The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.). When expressing a zalpha11 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25xc2x0 C. to 35xc2x0 C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto(trademark) Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto(trademark) yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
Mammalian cells suitable for use in assaying antagonist activity of the novel soluble receptors of the present invention express a zalpha11 receptor or receptor fusion capable of signaling and transducing a receptor-mediated signal of the zalpha11 Ligand. Such cells include cells that express a xcex2-subunit, such as gp130, IL-2Rxcex3 and cells that co-express receptors (Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat. No. 5,284,755). In this regard it is generally preferred to employ a cell that is responsive to other cytokines that bind to receptors in the same subfamily, such as IL-6 or LIF, because such cells will contain the requisite signal transduction pathway(s). Preferred cells of this type include the human TF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch et al., Blood 69:1782, 1987; Broudy et al., Blood 75:1622-1626, 1990). In the alternative, suitable host cells can be engineered to produce a xcex2-subunit or other cellular component needed for the desired cellular response. For example, the murine cell line BaF3 (Palacios and Steinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986) has been used to produce a cell line responsive to the zalpha11 Ligand (see Examples). Other such lines include a baby hamster kidney (BHK) cell line, or the CTLL-2 cell line (ATCC TIB-214) can be transfected to express an IL-2Rxcex3 subunit in addition to zalpha11 receptor. It is generally preferred to use a host cell and receptor(s) from the same species, however this approach allows cell lines to be engineered to express multiple receptor subunits from any species, thereby overcoming potential limitations arising from species specificity. In the alternative, species homologs of the human receptor cDNA can be cloned and used within cell lines from the same species, such as a mouse cDNA in the BaF3 cell line. Cell lines that are dependent upon one hematopoietic growth factor, such as IL-3, can thus be engineered to become dependent upon a zalpha11 ligand. Such cells can be used as described herein in the presence of zalpha11 Ligand to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 on zalpha11 Ligand signaling and proliferative activity.
Cells expressing functional soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 are used within screening assays. A variety of suitable assays are known in the art. These assays are based on the detection of a biological response in the target cell to the zalpha11 Ligand in the presence or absence of the soluble receptors of the present invention. One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of zalpha11 Ligand with or without the addition other cytokines or proliferative agents, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of Alamar Blue(trademark) (AccuMed, Chicago, Ill.) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay format uses cells that are further engineered to express a reporter gene. The reporter gene is linked to a promoter element that is responsive to the receptor-linked pathway, and the assay detects activation of transcription of the reporter gene. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989). Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. A preferred promoter element in this regard is a serum response element, or SRE (see, for example, Shaw et al., Cell 56:563-572, 1989). A preferred such reporter gene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987). Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al., J. Biol. Chem. 269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993). Luciferase assay kits are commercially available from, for example, Promega Corp., Madison, Wis. Target cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like for antagonist activity. Such cells can be used as described herein in the presence of zalpha11 Ligand in a competitive inhibition type assay to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 on zalpha11 Ligand signaling and proliferative activity.
T-and B-cell proliferation assay methods can also be used to assess the antagonist activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 on zalpha11 Ligand signaling and proliferative activity in the presence of other cytokines, for example, IL-15, Flt3 and the like. Such assays are described in the examples herein, and are know in the art. Briefly, using flow cytometry, mature or immature subsets of T-cells or B-cells are isolated based on the presence or absence of various cell surface molecules (e.g., CD4, CD8, CD 19, CD3, CD40, CD28, etc.). Cells can be selected prior to or after exposure to zalpha11 Ligand, depending on the cell type being studied, and the effect of zalpha11 Ligand thereon. The soluble receptors or antibodies of the present invention can be added at a range of concentrations to assess the antagonistic or binding activity on the Ligand in the T-cell or B-cell proliferation assay. Such assays are well known in the art, and described herein.
Moreover, a secretion trap method employing soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to isolate transfected cells that express zalpha11 Ligand. For the method, see, Aldrich, et al, Cell 87: 1161-1169, 1996. A cDNA expression library prepared from a known or suspected ligand source is transfected into COS-7 cells. The cDNA library vector generally has an SV40 origin for amplification in COS-7 cells, and a CMV promoter for high expression. The transfected COS-7 cells are grown in a monolayer and then fixed and permeabilized. Tagged or biotin-labeled soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, described herein, is then placed in contact with the cell layer and allowed to bind cells in the monolayer that express an anti-complementary molecule, i.e., a zalpha11 Ligand. A cell expressing a ligand will thus be bound with receptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 or anti-FLAG for FLAG-tagged fusions, streptavidin, and the like) which is conjugated with horseradish peroxidase (HRP) is used to visualize these cells to which the tagged or biotin-labeled soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides has bound. The HRP catalyzes deposition of a tyramide reagent, for example, tyramide-FITC. A commercially-available kit can be used for this detection (for example, Renaissance TSA-Direct(trademark) Kit; NEN Life Science Products, Boston, Mass.). Cells which express zalpha11 receptor Ligand will be identified under fluorescence microscopy as green cells and picked for subsequent cloning of the ligand using procedures for plasmid rescue as outlined in Aldrich, et al, supra., followed by subsequent rounds of secretion trap assay until single clones are identified.
Moreover, histologic and immunohistochemical methods employing soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to identify cells and tissues cells that express zalpha11 Ligand. Such methods are known in the art and described herein.
Additional assays to detect the antagonist or binding activity of soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides provided by the present invention include the use of hybrid receptor polypeptides. These hybrid polypeptides fall into two general classes. Within the first class, the intracellular domain of zalpha11, comprising approximately residues 256 (Lys) to 528 (Ser) of SEQ ID NO:2, is joined to the ligand-binding domain of a second receptor. It is preferred that the second receptor be a hematopoietic cytokine receptor, such as mpl receptor (Souyri et al., Cell 63:1137-1147, 1990). The hybrid receptor will further comprise a transmembrane domain, which may be derived from either receptor. A DNA construct encoding the hybrid receptor is then inserted into a host cell. Cells expressing the hybrid receptor are cultured in the presence of a ligand for the binding domain and assayed for a response. This system provides a means for analyzing signal transduction mediated by zalpha11 while using readily available ligands. This system can also be used to determine if particular cell lines are capable of responding to signals transduced by zalpha11. A second class of hybrid receptor polypeptides comprise the extracellular (ligand-binding) domain of zalpha11 (approximately residues 20 (Cys) to 237 (His) of SEQ ID NO:2) (SEQ ID NO:6) with a cytoplasmic domain of a second receptor, preferably a cytokine receptor, and a transmembrane domain. The transmembrane domain may be derived from either receptor. Such hybrid receptors are expressed in cells known to be capable of responding to signals transduced by the receptor comprising the extracellular domain, such as in the presence of the zalpha11 Ligand. Addition of the soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, in the presence of the zalpha11 Ligand, is used to assess the zalpha11 Ligand antagonist or binding activity of the soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides to the zalpha11 Ligand.
The tissue specificity and biological activities of zalpha11 Ligand expression suggest a role in early NK cell and thymocyte development, mature B-cell expansion, general immune response stimulation, and immune response regulation. These processes involve stimulation of cell proliferation and differentiation in response to the binding of the zalpha11 Ligand to its cognate receptor, comprising at least one zalpha11 receptor subunit. In view of the biological activity observed for this Ligand, antagonists have enormous potential in both in vitro and in vivo applications. As antagonists of the zalpha11 Ligand, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can find utility in the suppression of the immune system, such as in the treatment of autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, diabetes mellitus, inflammatory bowel disease, Crohn""s disease, and the like. Immune suppression can also be used to reduce rejection of tissue or organ transplants and grafts and to treat B-cell malignancies, T-cell specific leukemias or lymphomas by inhibiting proliferation of the affected cell type.
Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides may also be used within diagnostic systems for the detection of circulating levels of zalpha11 Ligand. Within a related embodiment, antibodies or other agents that specifically bind to soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides can be used to detect circulating receptor polypeptides. Elevated or depressed levels of Ligand or receptor polypeptides may be indicative of pathological conditions, including cancer. Soluble receptor polypeptides may contribute to pathologic processes and can be an indirect marker of an underlying disease. For example, elevated levels of soluble IL-2 receptor in human serum have been associated with a wide variety of inflammatory and neoplastic conditions, such as myocardial infarction, asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia, B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breast cancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).
A ligand-binding polypeptide of a zalpha11 receptor, such as soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 can be prepared by expressing a truncated DNA encoding the zalpha11 cytokine binding domain (approximately residue 20 (Cys) through residue 237 (His) of the human receptor (SEQ ID NO:2) (SEQ ID NO:6)) or the corresponding region of a non-human receptor (e.g., SEQ ID NO:12). A soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 can be prepared by co-expressing a truncated DNA encoding the zalpha11 cytokine binding domain (SEQ ID NO:6) and the truncated DNA encoding the extracellular domain of another class I cytokine receptor, such as IL-2Rxcex3 (SEQ ID NO:4). It is preferred that the extracellular domains of the soluble zalpha11 homodimer or heterodimer be prepared in a form substantially free of transmembrane and intracellular polypeptide segments. Moreover, ligand-binding polypeptide fragments within the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide (e.g., soluble zalpha11/IL-2Rxcex3), or cytokine-binding domain, described above, can also serve as zalpha11 soluble receptors for uses described herein. To direct the export of a receptor polypeptide from the host cell, the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide, secretory peptide from another cytokine receptor, other secreted molecule, or a zalpha11 receptor secretory peptide. To facilitate purification of the secreted receptor polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag(trademark) peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, Conn.), Glu-glu tag (SEQ ID NO:14) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the soluble receptor polypeptide.
In an alternative approach, a receptor extracellular domain can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an Fc fragment, which contains two constant region domains and lacks the variable region. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vitro assay tool, to block signals in vitro by specifically titrating out Ligand, and as antagonists in vivo by administering them parenterally to bind circulating ligand and clear it from the circulation. To purify ligand, a zalpha11-Ig chimera (e.g., Zalpha11-Fc4 described herein), is added to a sample containing the ligand (e.g., cell-conditioned culture media or tissue extracts) under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength). The chimera-ligand complex is then separated by the mixture using protein A, immobilized on a solid support (e.g., insoluble resin beads). The ligand is then eluted using conventional chemical techniques, such as with a salt or pH gradient. In the alternative, the chimera itself can be bound to a solid support, with binding and elution carried out as above. Collected fractions can be re-fractionated until the desired level of purity is reached.
Moreover, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3, can be used as a xe2x80x9cligand sink,xe2x80x9d i.e., antagonist, to bind ligand in vivo or in vitro in therapeutic or other applications where the presence of the ligand is not desired. For example, in cancers that are expressing large amounts of bioactive zalpha11 Ligand, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 can be used as a direct antagonist of the ligand in vivo, and may aid in reducing progression and symptoms associated with the disease, and can be used in conjunction with other therapies (e.g., chemotherapy) to enhance the effect of the therapy in reducing progression and symptoms, and preventing relapse. Moreover, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 can be used to slow the progression of cancers that over-express zalpha11 receptors, by binding ligand in vivo that would otherwise enhance proliferation of those cancers.
Moreover, soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 can be used in vivo or in diagnostic applications to detect zalpha11 Ligand-expressing cancers in vivo or in tissue samples. For example, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 can be conjugated to a radio-label or fluorescent label as described herein, and used to detect the presence of the zalpha11 Ligand in a tissue sample using an in vitro ligand-receptor type binding assay, or fluorescent imaging assay. Moreover, a radiolabeled soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 could be administered in vivo to detect Ligand-expressing solid tumors through a radio-imaging method known in the art.
It is preferred to purify the polypeptides of the present invention to xe2x89xa780% purity, more preferably to xe2x89xa790% purity, even more preferably xe2x89xa795% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
Expressed recombinant soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 (or zalpha11 chimeric or fusion polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles and Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be isolated by exploitation of their biochemical, structural, and biological properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, xe2x80x9cGuide to Protein Purificationxe2x80x9d, M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification. Moreover zalpha11 Ligand affinity columns can be used to purify soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3. Such affinity chromatography methods are well known in the art.
Moreover, using methods described in the art, polypeptide fusions, or hybrid zalpha11 proteins, are constructed using regions or domains of the inventive zalpha11 in combination with those of other human cytokine receptor family proteins, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
Soluble receptor fusion polypeptides or proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between zalpha11 of the present invention with the functionally equivalent domain(s) from another cytokine family member. Such domains include, but are not limited to, the extracellular cytokine binding domain, ligand binding domain and residues, transmembrane domain, , as disclosed herein. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zalpha11 polypeptide and those polypeptides to which they are fused. Generally, a DNA segment that encodes a domain of interest, e.g., a zalpha11 domain described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide (for instance a domain or region from another cytokine receptor, such as the IL-2Rxcex3 receptor), and inserted into an appropriate expression vector, as described herein. Generally DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof. For example, a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by a cytokine-binding domain. Such fusion proteins can be expressed, isolated, and assayed for activity as described herein.
Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 polypeptides, or fragments thereof may also be prepared through chemical synthesis. Such polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; and Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is with a reagent that cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art.
The activity of molecules of the present invention can be measured using a variety of assays that measure cell differentiation and proliferation. Such assays are well known in the art and described herein.
Proteins of the present invention are useful for example, in treating lymphoid, immune, hematopoietic, inflammatory disorders and the like, and can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For instance, host cells expressing a soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 can be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers are a means to entrap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic xe2x80x9cencapsulationsxe2x80x9d permit the diffusion of proteins and other macromolecules secreted or released by the captured cells to the recipient animal. Most importantly, the capsules mask and shield the foreign, embedded cells from the recipient animal""s immune response. Such encapsulations can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells.
The materials needed to generate the alginate threads are known in the art. In an exemplary procedure, 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5xc3x97105 to about 5xc3x97107 cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl2 solution over a time period of xcx9c15 min, forming a xe2x80x9cthreadxe2x80x9d. The extruded thread is then transferred into a solution of 50 mM CaCl2, and then into a solution of 25 mM CaCl2. The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer""s Solution and drawn from solution into a syringe barrel (without needle). A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer""s Solution.
Adenoviral and other viral systems, such as vaccinia virus can be used to express and produce the proteins of the present invention. For example, using adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential E1 gene has been deleted from the viral vector, and the virus will not replicate unless the E1 gene is provided by the host cell (the human 293 cell line is exemplary). If the adenoviral delivery system has an E1 gene deletion, the virus cannot replicate in human cells, but will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Moreover, by deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated. Generation of so called xe2x80x9cgutlessxe2x80x9d adenoviruses where all viral genes are deleted are particularly advantageous for insertion of large inserts of heterologous DNA. For review, see Yeh, P. and Perricaudet, M., FASEB J. 11:615-623, 1997.
The adenovirus system can be used for protein production in vitro. By culturing adenovirus-infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, are exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (See Garnier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
Soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 receptor antagonists can be used in vitro in an assay to measure a decrease in stimulation of colony formation by zalpha11 Ligand from isolated primary bone marrow cultures. Such assays are disclosed herein and are well known in the art.
Zalpha11 Ligand antagonists and binding agents are also useful as research reagents for characterizing sites of ligand-receptor interaction. Inhibitors of zalpha11 Ligand activity (zalpha11 Ligand antagonists) include anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric receptor polypeptide antibodues, such as anti-soluble zalpha11/IL-2Rxcex3 antibodies and soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).
A soluble zalpha11 receptor or soluble zalpha11 heterodimeric receptor polypeptides, such as soluble zalpha11/IL-2Rxcex3 ligand-binding polypeptide of the present invention, can also be used for purification of zalpha11 Ligand. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument may be advantageously employed (e.g., BIAcore(trademark), Pharmacia Biosensor, Piscataway, N.J.; or SELDI(trademark) technology, Ciphergen, Inc., Palo Alto, Calif.). Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
Ligand-binding receptor polypeptides, such as those of the present invention, can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949) and calorimetric assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).
Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides can also be used to prepare antibodies that bind to epitopes, peptides, or polypeptides contained within the antigen. The zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. One of skill in the art would recognize that antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides (e.g., SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:4). Polypeptides comprising a larger portion of a zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides i.e., from 30 to 100 residues up to the entire length of the amino acid sequence are included. Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein. Suitable antigens include the zalpha11 polypeptide encoded by SEQ ID NO:2 from amino acid number 20 (Cys) to amino acid number 237 (His) (SEQ ID NO:6), or a contiguous 9 to 218 AA amino acid fragment thereof. Preferred peptides to use as antigens are the cytokine binding domain, disclosed herein, and zalpha11 hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot, determined for example, from a Hopp/Woods hydrophilicity profile based on a sliding six-residue window, with buried G, S, and T residues and exposed H, Y, and W residues ignored. For example, zalpha11 hydrophilic peptides include peptides comprising amino acid sequences selected from the group consisting of: (1) amino acid number 51 (Trp) to amino acid number 61 (Glu) of SEQ ID NO:2; (2) amino acid number 136 (Ile) to amino acid number 143 (Glu) of SEQ ID NO:2; (3) amino acid number 187 (Pro) to amino acid number 195 (Ser) of SEQ ID NO:2; and (4) amino acid number 223 (Phe) to amino acid number 232 (Glu) of SEQ ID NO:2. The corresponding hydrophilic regions in reference to SEQ ID NO:6 can be made with cross-reference to the above amino acid residues of SEQ ID NO:2. Moreover, antigenic epitope-bearing polypeptides as predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.) are suitable antigens. In addition, conserved motifs, and variable regions between conserved motifs of zalpha11 soluble receptor are suitable antigens. Suitable antigens also include the zalpha11 polypeptides disclosed above in combination with another class I cytokine extracellular domain, such as those that form soluble zalpha11 heterodimeric polypeptides, such as soluble zalpha11/IL-2Rxcex3. Moreover, corresponding regions of the mouse soluble zalpha11 receptor polypeptide (residues 20 (Cys) to 237 (His) SEQ ID NO:12) can be used to generate antibodies against the soluble mouse zalpha11 receptor. In addition Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982.
As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide or a fragment thereof. The immunogenicity of a zalpha11 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund""s complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zalpha11 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is xe2x80x9chapten-likexe2x80x9d, such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term xe2x80x9cantibodiesxe2x80x9d includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(abxe2x80x2)2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally xe2x80x9ccloakingxe2x80x9d them with a human-like surface by replacement of exposed residues, wherein the result is a xe2x80x9cveneeredxe2x80x9d antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules. A threshold level of binding is determined if anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 antibodies herein bind to a soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3) polypeptide. It is preferred that the antibodies exhibit a binding affinity (Ka) of 106 Mxe2x88x921 or greater, preferably 107 M1 or greater, more preferably 108 Mxe2x88x921 or greater, and most preferably 109 Mxe2x88x921 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
Whether anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of a protein family. Screening can also be done using non-human soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, and soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 mutant polypeptides. Moreover, antibodies can be xe2x80x9cscreened againstxe2x80x9d known related polypeptides, to isolate a population that specifically binds to the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides. For example, antibodies raised to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 will flow through the matrix under the proper buffer conditions. Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically binding anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 antibodies can be detected by a number of methods in the art, and disclosed below.
A variety of assays known to those skilled in the art can be utilized to detect antibodies that bind to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 protein or polypeptide.
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 protein or peptide). Genes encoding polypeptides having potential binding domains for soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide, can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display libraries can be screened using the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 sequences disclosed herein to identify proteins which bind to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3. These xe2x80x9cbinding polypeptidesxe2x80x9d which interact with soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding polypeptides can also be used in analytical methods such as for screening expression libraries and neutralizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor. The binding polypeptides can also be used for diagnostic assays for determining circulating levels of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides; for detecting or quantitating soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides as marker of underlying pathology or disease. These binding polypeptides can also act as zalpha11 receptor or zalpha11 heterodimeric polypeptide, such as zalpha11/IL-2Rxcex3 xe2x80x9cantagonistsxe2x80x9d to block zalpha11 receptor or zalpha11 heterodimeric polypeptide, such as zalpha11/IL-2Rxcex3 binding and signal transduction in vitro and in vivo. Again, these anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 binding polypeptides would be useful for inhibiting zalpha11 Ligand activity, as well as receptor activity or protein-binding. Antibodies raised to the heterodimer or multimeric combinations of the present invention are preferred embodiments, as they may act more specifically against the zalpha11 Ligand, or more potently than antibodies raised to only one subunit. Moreover, the antagonistic and binding activity of the antibodies of the present invention can be assayed in the zalpha11 Ligand proliferation and other biological assays described herein.
Antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 may be used for tagging cells that express zalpha11 receptor or zalpha11 heterodimeric polypeptides, such as zalpha11/IL-2Rxcex3; for isolating soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptide by affinity purification; for diagnostic assays for determining circulating levels of soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides; for detecting or quantitating soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zalpha11 receptor or zalpha11 heterodimeric polypeptide, such as zalpha11/IL-2Rxcex3, or zalpha11 Ligand activity in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 or fragments thereof may be used in vitro to detect denatured or non-denatured soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
Antibodies to soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 are useful for tagging cells that express the corresponding receptors and assaying their expression levels, for affinity purification, within diagnostic assays for determining circulating levels of soluble receptor polypeptides, analytical methods employing fluorescence-activated cell sorting. Moreover, divalent antibodies, and anti-idiotypic antibodies may be used as agonists to mimic the effect of the zalpha11 Ligand.
Antibodies herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, antibodies or binding polypeptides which recognize soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 polypeptides of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (i.e., a zalpha11 receptor, or zalpha11 heterodimeric receptor, such as zalpha11/IL-2Rxcex3). More specifically, anti- soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the zalpha11 receptor or a zalpha11 heterodimeric receptor, such as zalpha11/IL-2Rxcex3 receptor molecules.
Suitable detectable molecules may be directly or indirectly attached to polypeptides that bind soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 (xe2x80x9cbinding polypeptides,xe2x80x9d including binding peptides disclosed above), antibodies, or bioactive fragments or portions thereof. Suitable detectable molecules include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Binding polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/anticomplementary pair, where the other member is bound to the binding polypeptide or antibody portion. For these purposes, biotin/streptavidin is an exemplary complementary/anticomplementary pair.
In another embodiment, binding polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, if the binding polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the fusion protein including only a single domain includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti-complementary-detectable/cytotoxic molecule conjugates.
In another embodiment, soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 binding polypeptide-cytokine or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood, lymphoid, colon, and bone marrow cancers), if the binding polypeptide-cytokine or anti-soluble zalpha11 receptor or anti-soluble zalpha11 heterodimeric polypeptide, such as anti-soluble zalpha11/IL-2Rxcex3 antibody targets the hyperproliferative cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). The described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable anti-zalpha11 homodimer and heterodimer antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), for instance.
Alternatively, soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 binding polypeptide or antibody fusion proteins described herein can be used for enhancing in vivo killing of target tissues by directly stimulating a zalpha11 receptor-modulated apoptotic pathway, resulting in cell death of hyperproliferative cells expressing zalpha11 receptor or a zalpha11 heterodimeric receptor, such as soluble zalpha11/IL-2Rxcex3 receptor.
Four-helix bundle cytokines that bind to cytokine receptors as well as other proteins produced by activated lymphocytes play an important biological role in cell differentiation, activation, recruitment and homeostasis of cells throughout the body. Therapeutic utility includes treatment of diseases that require immune regulation including autoimmune diseases, such as, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythomatosis and diabetes. Zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, may be important in the regulation of inflammation, and therefore would be useful in treating rheumatoid arthritis, asthma, ulcerative colitis, inflammatory bowel disease, Crohn""s disease, and sepsis. There may be a role of zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, in mediating tumorgenesis, and therefore would be useful in the treatment of cancer. Zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, may be a potential therapeutic in suppressing the immune system that would be important for reducing graft rejection. Soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 may have usefulness in prevention of graft vs. host disease.
Alternatively, zalpha11 Ligand antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptors in conjunction with other cytokines may enable selective activation, enhancement, or selective suppression, of the immune system in conjunction with zalpha11 Ligand on other cytokines which would be important in boosting immunity to infectious diseases, treating immunocompromised patients, such as HIV+ patient, or in improving vaccines. In particular, zalpha11 antagonists, including soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3, could prevent the expansion of a subset of the immune system involving zalpha11 Ligand (e.g., NK cells and mature B-cells), while enabling expansion of progenitors induced by other cytokines (e.g., T-cells), and would provide therapeutic value in treatment of viral infection and other infection. For example, with Dengue virus infection, which causes dengue hemorrhagic fever/Dengue Shock syndrome (DHF/DSS) it is believed that severe DHF/DSS occurs as a result of xe2x80x9cimmune enhancementxe2x80x9d i.e., enhanced replication of the virus in the presence of pre-existing antibodies against another serotype. In the second infection by a different Dengue virus serotype, the immune system raises antibodies against the first virus that cross-react but do not neutralize the virus, and that potentially aid its entry into macrophages. Thus, suppression of the antibody immune response, or B cell response, during a second or third Dengue infection may help the immune system react appropriately in the second infection to neutralize the virus by suppressing the xe2x80x9cenhancingxe2x80x9d antibodies from the first serotpye infection, and consequently avoiding severe DHF/DSS. For review, see White, D. O. and Fenner F. J. (Eds.) Medical Virology, 3rd ed., Academic Press, Orlando Fla., 1986, pages 479-508). Similarly, suppression of maternal antibody responses against fetal antigens by soluble receptors of the present invention can aid in preventing birth defects and spontaneous abortion. Moreover, in such applications the soluble receptors of the present invention can be used in conjunction with other cytokines to suppress some immune system activities (e.g., B-cell proliferation, using the soluble receptors) but allowing others to increase, e.g., in the presence of other cytokines described herein and known in the art.
The bioactive binding polypeptide or antibody conjugates described herein can be delivered orally, intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. For pharmaceutical use, the soluble zalpha11 receptor or soluble zalpha11 heterodimeric polypeptide, such as soluble zalpha11/IL-2Rxcex3 receptor polypeptides of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a zalpha11 soluble receptor polypeptide in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 xcexcg/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a therapeutically effective amount of zalpha11 soluble receptor polypeptide is an amount sufficient to produce a clinically significant effect.
The invention is further illustrated by the following non-limiting examples.